A large population of haploid Nicotiana tabacum plants was obtained from the F1 hybrid ‘LA Burley 21’ ✕ ‘Burley 21’ using an anther culture technique. LA Burley 21 is homoyzgous recessive (aabb) for total alkaloid content, and Burley 21 is homozygous dominant (AABB). Haploid genotypes for alkaloid content included AB, Ab, aB, and ab, obtained from the diploid F1 hybrid of genotype AaBb. The haploid plants were verified cytologically, and individual haploids were chromosomally doubled by either root or leaf in vitro tissue culture procedures to recover the four homozygous doubled haploid forms. The two parental genotypes (AABB and aabb) and the low‐intermediate (aaBB) and high‐intermediate (AAbb) genotypes were evaluated for stability and compared with conventionally derived alkaloid lines. Haploid plants were extracted from doubled haploid genotypes to obtain additional evidence on stability of anther‐derived haploids. The haploids and their derivatives behave as stable true‐breeding genotypes. The evidence presented supports the hypothesis that two major genes control total alkaloid production in burley tobacco. The feasibility of using the anther‐derived haploid technique for the development of tobacco breeding lines is demonstrated.